Multiple oligonucleotide containing oligomers and the cleanable linkers used in their preparation

ABSTRACT

A method for the synthesis of a plurality of oligonucleotides comprising the steps of 
     (i) forming a first oligonucleotide on a first cleavable link attached to a solid support; 
     (ii) attaching to the first oligonucleotide a cleavable linker moiety; 
     (iii) forming a second oligonucleotide on the cleavable linker moiety; and 
     (iv) cleaving the first cleavable link and the cleavable linker moiety to give a plurality of oligonucleotides; wherein the cleavable linker moiety is of the Formula (1): ##STR1## in which A 1 , A 2  and E are as defined herein, and novel compounds which may be used in the operation of the method.

This is a divisional of application Ser. No. 08/041,599, filed Apr. 5,1993 now U.S. Pat. No. 5,393,877.

This invention relates to a method for the synthesis of oligonucleotidesand to novel compounds which may be used during operation of the method.

Oligonucleotide sequences are routinely synthesised for use as linkers,adaptors, building blocks for synthetic genes, synthetic regulatorysequences, probes, primers and other purposes and a number of methodshave been developed for producing such sequences. These methods rely onthe initial attachment of a first suitably protected nucleoside to asolid support by a cleavable linkage followed by sequential reactions ofprecursors of individual nucleotides to the growing oligonucleotidestrand with each addition of a precursor involving a number of chemicalreactions. At present the method most generally employed for theproduction of a lone oligonucleotide is based on phosphoramiditechemistry. This is fully described by Caruthers et al in TetrahedronLetters 1981, 22, (20) pp 1859-62, by Koster et al in U.S. Pat. No.4,725,677 and by M. J. Gait (`Oligonucleotide Synthesis, a PracticalApproach`, IRL Press Oxford p 35-81).

Several types of automated DNA synthesisers are now commerciallyavailable which enable an oligonucleotide to be prepared usingphosphoramidite chemistry. An illustrative description of how a loneoligonucleotide may be formed by sequential reactions of precursors ofthe individual nucleotides on a support is provided in the protocol forthe Applied Biosystems DNA Synthesiser Model 380B, particularly Section2 thereof, which is incorporated herein by reference thereto.

In response to the rapid increase in demand for oligonucleotides,improvements are desirable which will increase the throughput ofcommercial synthesisers, i.e. increase the number of oligonucleotidessynthesised per day.

We have now developed a rapid and efficient method for the production ofoligonucleotides in which more than one oligonucleotide can besynthesised on the same support using a clearable linker moietyintroduced as required in a growing oligonucleotide chain.

According to a first aspect of the present invention there is provided amethod for the synthesis of a plurality of oligonucleotides comprisingthe steps of:

(i) forming a first oligonucleotide on a first cleavable link attachedto a solid support;

(ii) attaching to the first oligonucleotide a cleavable linker moiety;

(iii) forming a second oligonucleotide on the cleavable linker moiety;and

(iv) cleaving the first cleavable link and the cleavable linker moietyto give a plurality of oligonucleotides;

wherein the cleavable linker moiety is of the Formula (1): ##STR2##wherein one or both of A¹ and A² is a divalent group of the formula (a):##STR3## R¹ and R² are each independently H, optionally substitutedalkyl, optionally substituted alkoxy or optionally substituted aryloxy,halogen, cyano, nitro, optionally protected hydroxy, optionallyprotected oxycarbonyl, optionally protected NH₂, or an electronwithdrawing group; Y is CH₂, CH₂ CH₂, NH, S or O; E is an organic spacergroup; and any remaining group represented by A¹ or A² is of the formula(b): ##STR4## wherein n has a value of from 1 to 5; the carbon atommarked with an asterisk is attached to an oxygen atom shown in Formula(1); and each R³ independently represents H or optionally substitutedalkyl.

It is preferred that A¹ and A² are both of Formula (a) and n preferablyhas a value of from 1 to 3, more preferably 2.

The support is preferably a solid support such as is used in automatedoligonucleotide synthesis. The preferred solid support is, for example,a modified inorganic polymer such as those disclosed in the U.S. Pat.No. 4,458,066, a silica gel, Porasil C, kieselguhr PDMA, polystyrene,polyacrylamide, Silica CPG (LCAA) or a controlled pore glass as used in,for example, the Applied Biosystems DNA synthesiser Model 380B. Theoligonucleotides may be formed from precursors of individual nucleotidesby conventional technology used for synthesising oligonucleotides, forexample by using phosphoramidite chemistry on an automatedoligonucleotide synthesiser as described above. The firstoligonucleotide is preferably connected to the support by a hydrolysablegroup (e.g. a base labile group) as is known in the art.

The first aspect of the invention may be illustrated by the formation of2 oligonucleotides using different combinations of the phosphoramiditesof 2'-deoxyadenosine (dA), 2'-deoxyguanosine (dG), 2'-deoxycytidine(dC), and 2'-deoxythymidine (dT) separated by the cleavable linkermoiety of Formula (1) built up sequentially in a 3' to 5' direction fromthe 3' hydroxy of deoxyribose on a solid support according to the abovemethod described by M. J. Gait. After synthesis the sequence may berepresented by: ##STR5## wherein X is a cleavable link.

After cleaving the linker moiety ##STR6## and the link X by which thefirst oligonucleotide is attached to the solid support twooligonucleotides result: ##STR7##

Thus two oligonucleotides have been synthesised on a single solidsupport.

It is preferred that the clearable linker moiety connects the first andsecond oligonucleotides through a 3' and a 5' oxygen, more preferablythrough a phosphate, phosphite, phosphate ester, phosphite ester orH-phosphonate ester, one on each oligonucleotide.

The identity of the first cleavable link is not believed to be critical,it is preferably base labile, and may be for example any of thecleavable links used in automated oligonucleotide synthesisers, such asa link which contains a base labile ester group.

Organic residues of the cleaved linker moiety, such as hydrocarbonchains, preferably do not remain attached to the oligonucleotides aftercleavage step (iv) to avoid any adverse affects on the properties of theoligonucleotides which such residues can have.

The first aspect of the invention includes repetition of steps (ii) and(iii) any desired number of times, for example 1 to 100 times, orpreferably 1 to 5 times, to produce further oligonucleotides which areeach connected through a cleavable linker moiety. As will beappreciated, when steps (ii) and (iii) are repeated the furtheroligonucleotides are formed on the cleavable linker moiety attached tothe previously formed oligonucleotide and may be the same as ordifferent to the previously formed oligonucleotides.

The cleavable linker moiety or moieties may be cleaved by basehydrolysis to give a mixture of individual oligonucleotides which may,if required, be purified and separated. Any suitable base may be used,for example, aqueous ammonia, methylamine.

In this specification the term "oligonucleotide" includes anoligodeoxyribonucleotide, an oligoribonucleotide and analogues thereof(for example those which bear protecting groups), including those withmethyl-phosphonate and phosphorothioate or phosphorodithioate diesterbackbones, and oligonucleotides with oligodeoxyribonucleotides,especially the 2'-oligodeoxyribonucleotides being more usuallysynthesised by the method of the invention. The preferredoligonucleotides are oligodeoxyribonucleotides, are essentially singlestranded, and are preferably from at least two, more preferably at least5, especially from 10 to 200 bases long.

To users of DNA synthesisers the method of the invention gives theadvantage of more effective use of the apparatus and subsequentlyreducing the cost of production and purification of oligonucleotides.

Thus with the present invention, the DNA synthesiser can produce two ormore oligonucleotides (which may be the same or different) on any one ofits columns without being re-programmed between each oligonucleotide.Thus when synthesis of one oligonucleotide is completed at a timeoutside the working day the synthesiser can go on to produce anotherwithout any intervention by an operative. This can significantlyincrease the productivity of such apparatus.

The method of the invention is particularly useful for the synthesis ofprimers for the Polymerase Chain Reaction (PCR) technique. At present alarge proportion of oligonucleotides synthesised are for this purpose.Such primers are typically required in pairs and the method of theinvention is convenient since it allows production of oligonucleotidesin pairs. This is particularly an advantage when using single columnsynthesisers and/or for heavily used facilities for out-of hoursworking.

It is preferred that the precursors of the individual nucleotides arenucleoside phosphoramidites protected, at the 5' oxygen atom andoptionally are base protected. Methods of protecting nucleoside basesare known in the art, for example by a protecting group which isremovable by treatment with mild acid or alkali. Adenine and cytosinemay be protected by an optionally substituted N-benzoyl group andGuanine by an N-isobutyryl group. Thymine and uracil generally do notrequire protection. Adenine and guanine may also be protected by adimethylformamide or phenoxyacetyl group, and cytosine by an isobutyrylgroup. The protecting groups are preferably removed after separation ofthe protected oligonucleotide from the support. Cleavage of the linkedmoiety may be effected before, during or after the removal of theprotecting groups depending upon the chemistry employed. It is preferredthat the protecting groups are removable by treatment with aqueous base,particularly concentrated ammonia solution or methylamine. In apreferred embodiment of the invention the cleavable linker moiety iscleavable under basic or alkaline conditions so that protecting groupremoval and cleavage of linker moieties can be effected in one step.Typical basic conditions employed, are to mix the protectedoligonucleotide with base, for example, concentrated aqueous ammonia,methylamine or a mixture of both, or for example, methanolic sodiumhydroxide. The reaction may be carried out, for example at a temperatureof room temperature (20° C.) to 100° C., more preferably from 50° to 90°C., especially around 55° to 60° C. The reaction is typically carriedout over a period of 48 hours, more particularly over a period up to 24hours, especially from about 5 to 24 hours. It is preferred that alinker moiety is chosen such that cleavage is completed under theseconditions.

Other bases, preferably volatile bases may be employed to effectcleavage. These may conveniently be organic amines in water, for examplepiperidine, diethylamine, or triethylamine, preferably at aconcentration from 10-70%.

As examples of precursors of individual nucleotides suitable for use inthe method there may be mentioned the2-cyanoethyl-N,N-diisopropylaminophosphoramidites of5'-dimethoxytrityl-N-4-benzoyl-2'-deoxycytidine,5'-dimethoxytrityl-N-2-isobutyryl-2'-deoxyguanosine,5'-dimethoxytrityl-N-6-benzoyl-2'-deoxyadenosine, and5'-dimethoxytritylthymidine.

For the synthesis of oligoribonucleotides precursors are, for example,the same as for oligodeoxyribonucleotides except that on the 2' positionof the ribose there is a protected hydroxyl group, for example atertiary butyl dimethyl silyloxy group or 2'--O--Fpmp phosphoramiditesfrom Cruachem Ltd.

There is an increasing interest in the use of oligonucleotides having a5' phosphate group (see e.g. Higuchi & Ockman (1989), Nucl. Acid Res.17(14), p5865). Therefore a synthetic method that gives rise to anoligonucleotide having a 5' phosphate group is of value.

Thus, in a further aspect the method of the present invention step (iv)yields desired oligonucleotides each having a group selected fromhydroxy and phosphate at the 3' and 5' position.

The cleavable linker moiety of Formula (1) may be attached to the firstoligonucleotide using a reagent of Formula (3) in place of a precursorof an individual nucleotide in a manner analogous to conventionaloligonucleotide synthesis.

Accordingly the present invention provides a compound of Formula (3):##STR8## wherein A¹, A² and E are as hereinbefore defined; Z¹ is an acidlabile protecting group; and --O--PA is a phosphoramidite group, aphosphate ester group or a H-phosphonate group.

Suitable acid labile protecting groups represented by Z¹ will beapparent to those skilled in the art and include those discussed in`Protective Groups in Organic Synthesis` by T. W. Greene, WileyInterscience. Examples of such protecting groups include methoxytrityl(preferably for oligoribonucleotide synthesis only), dimethoxytritiyl,pixyl, isobutyloxycarbonyl, t-butyl dimethylsilyl and like protectinggroups. Preferably, Z¹ is dimethoxytrityl.

E is preferably an organic spacer group having a length of 2 to 15, morepreferably 2 to 6 carbon atoms. E is preferably an alkyl, alkenyl, arylor aralkyl spacer group, optionally interrupted by an ether, thioether,amino or amido group. Preferred groups represented by E are optionallysubstituted phenylene, C₂₋₆ -alkylene, more preferably --CH₂ CH₂ --.

R¹ and R² are preferably each independently H; straight or branchedchain C₁₋₆ -alkyl, especially methyl, ethyl, propyl, butyl or tertiarybutyl; or C₁₋₆ -alkoxy, especially methoxy, ethoxy, propoxy and butoxy,particularly methoxy; halogen, cyano, nitro, phenoxy, optionallyprotected hydroxy; optionally protected oxycarbonyl; or optionallyprotected NH₂.

Y is preferably CH₂, CH₂ CH₂, NH or O, more preferably CH₂, CH₂ CH₂ orO, especially O.

Each R³ independently is preferably C₁₋₆ -alkyl, especially methyl,ethyl, propyl or butyl or, more preferably, H. Optional substituents areas defined for R¹ and R².

As examples of groups represented by formula (b) there may be mentioned--*CH₂ CH₂ --SO₂ --CH₂ --CH₂ -- and --*CHCH₃ --CH₂ --SO₂ --CH₂ --CH₂ --.

As will be understood, when --O--PA is a H-phosphonate or aphosphoramidite these are oxidised to respectively a phosphate diesteror phosphate tri-ester groups during operation of the method, forexample using aqueous iodine or peroxide. In the case of H-phosphonatethe oxidation is preferably performed after step (iii) and before step(iv), whilst in the case of phosphoramidite it is preferably performedduring step (i) and step (iii).

As examples of phosphate ester groups and H-phosphonate groups there maybe mentioned groups which, in the free acid form, are respectively offormula: ##STR9## wherein Z² is a protecting group, preferably a baselabile protecting group, for example 2-chlorophenyl or2,4-dichlorophenyl.

Preferably, --O--PA is a phosphoramidite of general structure: ##STR10##wherein R₅ and R₆ are each independently optionally substituted alkyl,especially C₁₋₄ -alkyl; optionally substituted aralkyl, especiallyoptionally substituted benzyl; cycloalkyl and cycloalkylalkyl containingup to ten carbon atoms, such as cyclopentyl or cyclohexyl; or R₅ and R₆taken together with the nitrogen atom to which they are attached form anoptionally substituted pyrollidine or piperidine ring or R₅ and R₆ whentaken together with the nitrogen atom to which they are attached form asaturated nitrogen heterocycle which optionally includes one or moreadditional hetero atom from the group consisting of nitrogen, oxygen andsulphur. R₅ and R₆ are preferably iso-propyl.

R₇ represents a hydrogen atom or a protecting group, for example aphosphate protecting group. As examples of phosphate protecting groupsthere may be mentioned optionally substituted alkyl groups, for examplemethyl, 2-cyanoethyl, 2-chlorophenyl, 2,2,2-trihalo-1,1-dimethyl ethyl,5-chloroquin-8-yl, 2-methylthioethyl and 2-phenylthioethyl groups inwhich the phenyl ring is optionally substituted, for example by a groupselected from halogen, eg. chlorine, or NO₂. Preferably R₇ is methyl or,more preferably, 2-cyanoethyl.

As will be appreciated by the skilled person the compounds of thepresent invention can exist in either the cis or trans form. However,the trans form of the compounds demonstrate a slower rate of cleavageand therefore result in oligonucleotides bearing terminal organicphosphate groups which may find applications in situations where thetermini are required to be blocked.

The compounds of Formula (3) are suitable reagents for attaching acleavable linker moiety of formula ##STR11## between a first and secondoligonucleotide as described by the method of the invention. Undersuitable conditions, for example treatment with concentrated ammoniumhydroxide, the compounds cleave to give the desired oligonucleotidesfree from any organic residue of the compound of Formula (3). This is ofparticular value where oligonucleotides are desired with free orphosphated 3' or 5' termini.

The utility of compounds of Formula (3) can be illustrated by referenceto the preparation of the sequence of Formula (2) as discussed above.For example, when A² is of Formula (a) the oligonucleotide of formulad(AGCTA) results having a 5'--OH group, and when A² is of Formula (b)d(AGCTA) results having a 5'-phosphate group. Accordingly, byappropriate selection of A² in a compound of Formula (3) from (a) and(b) the method of the invention provides the great benefit of enablingone to select whether the first, second, and subsequent oligonucleotidesprepared according to the method of the invention have a hydroxy groupat the 3' position and a hydroxy or phosphate group at the 5' position.

Compounds of Formula (3) wherein --O--PA is a phosphoramidite may beprepared by reacting a compound of the formula ##STR12## with a compoundof formula X¹ -PA in CH₂ Cl₂ using di(N-isopropyl)ethylamine as base. PAis preferably a phosphoramidite as defined above for --O--PA except that--O-- is absent, and Z¹, A¹, E and A² are as hereinbefore defined, andX¹ is a leaving group, for example Cl or Br.

When --O--PA in Formula (3) is a phosphate ester group as hereinbeforedefined the compound of Formula (3) may be prepared by reaction of acompound of formula ##STR13## with the triazolide of the correspondingfree phosphate ester using a method analogous to that described in theabove book by M. J. Gait.

When --O--PA in Formula (3) is a H-phosphonate group as hereinbeforedefined the compound of Formula (3) may be prepared by reaction in acompound of formula ##STR14## with PCl₃ in the presence of1,2,4-triazole using a method analogous to that described by B. C.Froehler et al, Nucleic Acid Research, (1986), 14, 5399-5407.

The compound of formula ##STR15## may be prepared, for example, byreaction of a compound of formula ##STR16## with a compound of formulaHO--A² --OH, preferably in an aprotic solvent using a suitablecondensing agent such as the aforementioned DCCI or1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide.

The compound of formula ##STR17## may be prepared by the reaction of thecompound of formula Z¹ --O--A¹ --OH with an activated form of thecompound of formula HO₂ C--E--CO₂ H, preferably in an aprotic solvent inthe presence of a molar equivalent of base. The dicarboxylic acid may beactivated to attack by the hydroxyl group by,being present as the acidanhydride, the acid chloride or;some other suitable derivative, or thereaction may be mediated by the presence of a coupling agent asdescribed above.

The compound of formula Z¹ --O--A¹ --OH may be prepared by the reactionof the compound of formula HO--A¹ --OH with Z¹ --Cl (or some othersuitably activated form of Z¹) in an anhydrous aprotic solvent in thepresence of a molar equivalent of base.

When A¹ is of Formula (a) shown above the compound of formula Z¹ --O--A¹--OH is preferably prepared by debenzoylation of a compound of formulaZ¹ --O--A¹ --O--CO--Ph in methanol using methylamine. The compound offormula Z¹ --O--A¹ --O--CO--Ph may be prepared by reaction of compoundof formula HO--A¹ --O--CO--Ph with Z¹ --X¹, wherein X¹ is a leavinggroup, for example Cl.

In the above processes for the preparation of the compound of Formula(3), and precursors thereof, Z¹, A¹, E, A², O, PA and Z² are ashereinbefore defined and DCCI is 1,3-dicylohexylcarbodiimide.

According to a further aspect of the invention there is provided acompound comprising two or more oligonucleotides linked, preferably by3' and 5' oxygen atoms, by a group or groups containing a cleavablelinker moiety of formula ##STR18## wherein A¹, E and A² are ashereinbefore defined. It is preferred that the cleavable linker moietyof formula ##STR19## is connected to each oligonucleotide via aH-phosphonate, phosphate, phosphite, phosphate ester or phosphite esterlinkage. It is preferred that one of the oligonucleotides is connectedto a support.

H-phosphonate linkages are of formula --HP(═O)--, preferred phosphiteester linkages are of formula --P(═O)--OR₆.-- and preferred phosphatelinkages are of formula --P(--OR₆)-- wherein R₆ is as hereinbeforedefined.

The invention is illustrated by the following non-limiting examples:

EXAMPLE 1

Preparation of Reagent A1

This was synthesised using the preparation numbered 1 to 4 describedbelow. DMT is 4,4'-dimethoxytrityl.

Step 1--Preparation of: ##STR20##

To a solution of 1,4-anhydroerythritol (10.4 g, 100 mmol) in drypyridine (100 ml) at 0° C. benzoyl chloride (14 g, 100 mmol) was addeddropwise with stirring. When addition was complete, the solution wasallowed to warm to room temperature and stirring was maintained for afurther two hours. To this solution was added 4,4'-dimethyoxytritylchloride (37.4 g, 110 mmol) and 4-(N,N-dimethylamino)pyridine (100 mg)and the mixture was stirred at room temperature for 16 hours. Thesolvent was removed by rotary evaporation and the residue redissolved indichloromethane and washed three times with saturated sodium bicarbonatesolution. The dichloromethane solution was dried by the addition ofanhydrous sodium sulphate and filtered. The filtrate was evaporated to agum and redissolved in methanol saturated with methylamine. Theresultant solution was incubated at room temperature until no startingmaterial could be detected by TLC. The solvent was removed by rotaryevaporation, and the residue redissolved in the minimum volume ofdichloromethane/methanol (9/1) and loaded on a silica chromatographycolumn. Elution with the same solvent gave the title compound as a whitefoam (22 g, 54%). ¹ H NMR: (δ, CDCl₃): 3.35, 1H, multiplet, CHOH; 3.5,2H, doublet, --CH₂ --; 3.75, 8H, complex multiplet, 2×--OCH₃ and --CH₂--; 4.2, 1H, complex multiplet, DMT--OCH; 6.9, 4H, complex multiplet,aromatics; 7.25-7.5, 9H, complex multiplet, aromatics.

Step 2--Preparation of: ##STR21##

The product from step 1 (10 g, 24.6 mmol) was dissolved in dry pyridine(150 ml) and succinic anhydride (10 g, 100 mmol) was added. Whendissolution was complete, 4-(N,N-dimethylamino)pyridine (500 mg) wasadded and the solution was stirred at room temperature overnight. Thesolvent was evaporated under reduced pressure and residual pyridineremoved by repeated co-evaporation with toluene. The residue wasredissolved in dichloromethane (500 ml) and washed three times with icecold 10% citric acid solution. The organic layer was separated, dried(sodium sulphate) filtered and evaporated to a gum which was redissolvedin the minimum volume of dichloromethane/methanol (9/1) and loaded on asilica chromatography column. Elution with the same solvent gave thetitle compound as a white foam (9.7 g, 78%).

¹ H NMR: (δ, CDCl₃): 2.75, 4H, multiplet, 2×COCH₂ ; 2.9 and 3.2, 2H, twopseudo triplets, --CH₂ --; 3.75, 8H, complex multiplet, 2×--OCH₃ and--CH₂ --; 4.2, 1H, complex multiplet, DMT--OCH; .5.0, 1H, multiplet,CHOCO; 6.9, 4H, complex multiplet, aromatics; 7.25-7.5, 9H, complexmultiplet, aromatics.

Step 3--Preparation of: ##STR22##

The product from step 2 (10 g, 19.5 mmol) was dissolved in dry pyridine(200 ml) containing 1,4-anhydroerythritol (10.4 g, 100 mmol). To thissolution was added 1-(3-dimethylaminopropyl)-3-ethyl carbodiimidehydrochloride (Aldrich, 3.75 g, 19.5 mmol). The solution was stirred atroom temperature overnight when TLC in dichloromethane:methanol (19:1)showed there to be no starting material present. The solvent was removedunder reduced pressure and residual pyridine removed by repeatedco-evaporation with toluene. The residue was redissolved in ethylacetate and washed three times with saturated sodium bicarbonatesolution and once with water. The organic layer was separated, dried(sodium sulphate), filtered and evaporated under reduced pressure togive a gum which was redissolved in the minimum volume ofdichloromethane:methanol (19:1) and loaded on a silica chromatographycolumn. Elution with the same solvent gave the title compound as acolourless gum (9.5 g, 79%).

¹ H NMR: (δ, CDCl₃): 2.75-2.9, 5H, multiplet, 2×COCH₂ +--OH; 3.2, 2H,multiplet, --CH₂ --; 3.65-3.85, 10H, complex multiplet, 2×--OCH₃ and2×--CH₂ --; 3.9-4.1, 2H, multiplet, --CH₂ --; 4.2, 1H, complexmultiplet, DMT--O--CH; 4.4, 1H, multiplet, CHOH; 5.0 and 5.1, 2H, twomultiplets, 2×CHOCO; 6.9, 4H, complex multiplet, aromatics; 7.25-7.5,9H, complex multiplet, aromatics.

Step 4--Preparation of Reagent A1 ##STR23##

The product from step 3 (2 g, 3.3 mmol) was dissolved in drydichloromethane (50 ml) and the solution stirred under a stream of dryargon. To this Solution was added dry diisopropylethylamine (2.7 ml, 16mmol) and 2-cyanoethyl-N,N-diisopropylaminochlorophosphine (1.05 ml,4.72 mmol). The solution was stirred at room temperature under a streamof dry argon for 30 minutes when TLC in dichloromethane:triethylamine(19:1) showed there to be no starting material present. The reaction wasquenched by addition of dry methanol (5 ml) and the solution was dilutedwith ethyl acetate (200 ml). This solution was washed with three equalvolumes of saturated sodium chloride solution, and one volume of water.The organic layer was separated, dried (sodium sulphate), filtered andevaporated under reduced pressure to a gum which was redissolved in theminimum volume of dichloromethane:triethylamine (19:1) and loaded on asilica choromatography column. Elution with the same solvent gave thetitle reagent Al as a colourless gum (1.8 g, 65%).

¹ H NMR: (δ, CDCl₃): 1.1-1.3, 12H, multiplet, 4×CH₃ --; 2.6, 2H,multiplet, CH₂ CN; 2.8-3.2, 6H, multiplet, 2×COCH.sub. 2 and --CH₂ --;3.5-3.9, 14H, complex multiplet, 2×--OCH₃, 2×CH(CH₃), POCH₂ --, 2×--CH₂--; 4.0-4.2, 3H, complex multiplet, --CH₂ and DMT--OCH; 4.5, 1H,multiplet, CH--O--P; 5.0, 1H, multiplet, CHOCO; 5.3, 1H, multiplet,CHOCO; 6.8, 4H, multiplet, aromatics; 7.2-7.5, 9H, complex multiplet,aromatics.

EXAMPLE 2

Preparation of two oligonucleotides on a single support

An oligonucleotide was formed on a solid support via a first(conventional) cleavable link using the protocol supplied with theApplied Biosystems 380B DNA synthesiser, using the3'-(2-cyanoethyl)-N,N-diisopropylaminophosphoramidites of5'-dimethoxytrityl-N⁴ -benzoyl-2'deoxycytidine, 5'-dimethoxytrityl-N²-isobutyryl-2'deoxyguanosine, 5'-dimethoxytrityl-N⁶-benzoyl-2'deoxyadenosine and 5'-dimethoxytritylthymidine (Cruachem) asthe precursors of the individual nucleotides. A cleavable linker moietywas attached to the first oligonucleotide by means of reagent A1.Reagent A1 was dissolved in anhydrous acetonitrile to a concentration of0.1M, and a bottle containing this solution was attached to one of thespare reagent ports on the DNA synthesiser. A column containingcontrolled pore glass or solid support bearing a 5'-protected nucleoside(in this case deoxyadenosine) connected by means of a (conventional)cleavable link (succinylglycylglycylaminopropyl, Cruachem) was attachedto the synthesiser. The synthesiser was then programmed to synthesisethe following sequence (SEQ ID NO:1):

    (5') TTTTTTTTTT--L'--TCGA (3')

(whereby the cleavable linker moiety L' is introduced by means ofreagent A1), using standard Synthesis cycles employed on the AppliedBiosystems 380B DNA synthesiser. The duration of the reaction steps andthe volume of reagents used for coupling, oxidation, capping anddetritylation were identical for each coupling, including that ofreagent A1. The synthesiser was programmed to perform the conventionalconcentrated ammonia wash of the column to release the oligonucleotidesinto collection vials.

In this manner the synthesiser achieves the steps of a) forming a firstoligonucleotide of sequence (5'-3') TCGA by succesive reaction of thenucleotide precursors with an oligonucleotide connected to thecontrolled pore glass support via the 3'--OH group and a (conventional)first cleavable link, b) attaching to the first oligonucleotide acleavable linker moiety by means of reagent A1, and c) forming a secondoligonucleotide on the clearable linker moiety having the sequence(5'-3') TTTTTTTTTT, to give two oligonucleotides separated by acleavable linker moiety and bound to a solid support by a cleavablelink, as illustrated by the formula: ##STR24## wherein --L'-- is acleavable linker of formula: ##STR25## attached to the 5' and 3' oxygenof the first and second oligonucleotides respectively by a group offormula --P(O)(OCH₂ CH₂ CN)--O--, and X is a first cleavable linkcontained in the succinylglycylglycylaminopropyl spacer. The synthesiseralso performs the cleavage of the first cleavable link X by the ammoniumtreatment as in step d) in the method of the invention.

To the vial containing the eluted oligonucleotide in the ammoniumsolution was added 1 ml of 40% aqueous methylamine, and the vial wasthen incubated at 55° C. for 16 hours and evaporated to dryness underreduced pressure. The residue was redissolved in 1 ml of water.

Five other oligonucleotides were also synthesised by conventionalprocedures as described above but omitting the treatment withmethylamine. These were designed to represent control molecules to beused in the analysis of products generated by the methylamine treatmentabove. These oligonucleotides had the following sequences:

1) (5') TTTTTTTTTT (3')

2) (5') TCGA (3')

3) (5') PO₄ ²⁻ TTTTTTTTTT (3')

4) (5') PO₄ ²⁻ TCGA (3')

5) (5') TTTTTTTTTTTTCGA (3') (SEQ ID NO:2)

Thus, oligonucleotides 1) and 2) are of identical length and sequence tothe products expected from cleavage of the oligonucleotide containingthe clearable link, and oligonucleotides 3, 4 and 5 are representativeof the products expected from partial cleavage of the oligonucleotidecontaining the clearable link.

The mixture of oligonucleotides produced in step d) described above wasanalysed by ion-exchange HPLC on a Pharmacia Mono-Q column using alinear gradient from 0-25% buffer B in buffer A over 35 minutes wherebuffer A was 50 mM Tris-chloride (pH 7.5) and buffer B was 50 mMTris-chloride/800 mM sodium chloride (pH 7.5).

Under these conditions, control oligonucleotide 1) had a retention timeof 29 minutes, control sequence 2) had a retention time of 9 minutes,control sequence 3) had a retention time of 32 minutes, control sequence4) had a retention time of 13 minutes and control sequence 5) had aretention time of 39 minutes.

The HPLC profile of the mixture of oligonucleotides produced in step d)above showed only peaks corresponding to the presence ofoligonucleotides 1 and 2, thus confirming that complete scission of thecleavable link had occurred, generating the desired products.

EXAMPLE 3

Preparation of Reagent A2

This was synthesised using the preparations numbered 1 to 2 below. DMTis 4,4'-dimethoxytrityl.

Step 1: Preparation of: ##STR26##

The product from Example 1), step 2) (10 g, 19.5 mmol) was dissolved indry pyridine (200 ml) containing 2,2'-sulfonyldiethanol (15 g, dried byazeotropic distillation with toluene below 45° C.). To this solution wasadded 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (3.75g, 19.5 mmol) and the whole was stirred at room temperature overnight.The solvent was removed under reduced pressure and residual pyridine wasremoved by repeated co-evaporation with toluene. The gummy residue wasredissolved in ethyl acetate (300 ml) and washed with saturated sodiumbicarbonate (3×200 ml) and water (200 ml). The organic layer wasseparated, dried (MgSO₄), filtered and evaporated to give a gum whichwas redissolved in the minimum volume of dichloromethane:methanol (19:1)and applied to a silica column. Elution with the same solvent gave thetitle compound as a colourless gum (8.2 g, 66.3%).

¹ H NMR δ (CDCl₃): 2.75 (4H, m, 2×COCH₂); 2.9-3.3 (4H, m, --CH₂ -- and--CH₂ OH); 3.45 (2H, t, CH₂ SO₂); 3.7-3.9 (8H, m, 2×--OCH₃ and --CH₂--); 4.0 (2H, t, CH₂ SO₂); 4.2 (1H, m, DMT--OCH); 4.55 (2H, t, --CH₂OCO); 5.0 (1H, m, CHOCO); 6.9 (4H, m, aromatics); 7.25-7.5 (9H, m,aromatics).

Step 2: Preparation of reagent A2: ##STR27##

The product from Step 1) (4 g, 6.2 mmol) was dissolved in drydichloromethane (50 ml) containing dry diisopropylethylamine (4.4 ml, 25mmol) and the solution was stirred under a stream of dry argon while2-cyanoethyl-N,N-diisopropylaminochlorophosphine (1.67 ml, 7.44 mmol)was added dropwise. The solution was stirred under argon at roomtemperature for a further thirty minutes when TLC indichloromethane:triethylamine (19:1) showed there to be no startingmaterial present. The reaction was quenched by the addition of drymethanol (5 ml), and the solution was diluted with ethyl acetate (200ml). This solution was washed with brine (3×200 ml) and water (200 ml).The organic layer was separated, dried (MgSO₄), filtered and evaporatedto a gum which was redissolved in the minimum volume ofdichloromethane:hexane:triethylamine (42:55:3) and applied to a silicacolumn. Elution with the same solvent followed by elution withdichloromethane:triethylamine (19:1) gave the title compound as acolourless glass (3.8 g, 72.5%)

¹ H NMR: δ (CDCl₃): 1.15-1.3 (14H, m, 2×CH(CH₃)₂); 2.6-2.75 (6H, m,2×COCH₂ and CH₂ CN); 3.3 (2H, m, --CH₂ --); 3.45-3.6 (6H, m, CH₂ SO₂ and2×CH₂ OP); 3.7-4.0 (10H, m, 2×--OCH₃, --CH₂ -- and --CH₂ SO₂); 4.2 (1H,m, DMT--OCH); 4.5 (2H, m, --CH₂ OCO); 5.0 (¹ H, m, CHOCO); 6.9 (4H, m,aromatics); 7.2-7.5 (9H, m, aromatics).

EXAMPLE 4

The method of Example 2) was repeated to synthesise twooligodeoxyribonucleotides bound to a solid support, except that ReagentA2 was used, dissolved in anhydrous acetonitrile to a concentration of0.1M, in place of Reagent A1.

The two oligonucleotides bound to a solid support by a cleavable linkare illustrated by the formula given in Example 2), wherein --L'-- is acleavable linker of formula: ##STR28##

The two oligonucleotides found after step d) in the method of theinvention were found to be identical to oligonucleotides 1) and 4)described in Example 2), demonstrating scission of the cleavable linkand phosphorylation of the 5'-hydroxyl of the oligonucleotide formed instep a) in the method of the invention.

EXAMPLE 5

Step 1--Preparation of Reagent A3

This was synthesised using the preparations numbered a) to f) below. DMTis 4,4'-dimethoxytrityl.

a) Preparation of r-3, Cis-4-dihydroxy-cis plustrans-2,-trans-5-dimethoxytetrahydrofruan ##STR29##

2,5-Dihydro-2,5-dimethoxyfuran (50 g; Aldrich, cis/trans mixture) wasdissolved in tetrahydrofuran (500 ml) in a 5 liter, three-necked flaskfitted with a mechanical stirrer. The contents of the flask were cooledto -5° C., and a solution of potassium permanganate (61.9 g) in water(2250 ml) was added dropwise with vigorous stirring at such a rate as tokeep the temperature of the flask contents between 4° and 6° C. Thisaddition took 80 minutes. The reaction mixture was then left stirringand allowed to warm to room temperature over 15 hours. The precipitatedmanganese dioxide was filtered off through Celite and washed with THF(200 ml). The clear colourless filtrate was evaporated and the residuewas shaken vigorously with ethyl acetate (200 ml) until no materialadhered to the flask wall. The fine precipitate of KOH was filtered offon a glass sinter and washed with ethyl acetate. The filtrate was rotaryevaporated to give the title compound as a golden syrup (24.4 g, 38.6%).The compound was mainly the meso product (80%) contaminated with the dlcompound (20%).

13C-NMR: meso product CH₃ O 55.462 ppm CHOH 75.303 ppm CHOCH₃ 109.606ppm dl product CH₃ O56.451 ppm CHOH 70.654 and 73.604 ppm CHOCH₃ 103,155and 108.257 ppm

b) Preparation of ##STR30##

The product from step a) above (1.7 g) was dissolved in dry pyridine (10ml) and 4-N,N-dimethylaminopyridine (DMAP) (100 mg) was added. Themixture was swirled to dissolve the solid and benzoyl chloride (1.2 ml)was added. The mixture was then left to stand at 20° C. for 16 hours.The mixture was rotary evaporated, and residual pyridine was removed byrepeated co-evaporation with toluene. The residual oil was partitionedbetween ethyl acetate and 1M HCl (40 ml of each). The organic layer waswashed in succession with water, 1M NaHCO₃, and saturated brine (40 mlof each), dried over magnesium sulphate, filtered and rotary evaporated.The residual oil was redissolved in dichloromethane/methanol (19/1) andapplied to a silica column. Elution with the same solvent gave thedesired product as a white solid (1.11 g, 38%)

¹³ C-NMR: CH₃ O: 55.680 and 55.951 ppm CHOH and CHOCO: 75.016 and 77.973ppm CHOCH₃ : 107.136 and 109.899 ppm C₆ H₅ : 128.576, 129.300, 129.957and 133.952 ppm CO: 166.076 ppm

c) Preparation of ##STR31##

The product from step b) above (2.47 g), 4,4'-dimethoxytrityl chloride(3.12 g) and DMAP (0.15 g) were stirred in dry pyridine (20 ml) at roomtemperature for 16 hours. The solvent was removed by rotary evaporationand residual pyridine was removed by repeated co-evaporation withtoluene. The residue was partitioned between ethyl acetate and water (50ml of each) and the organic layer was washed with 1M sodium bicarbonate,then saturated brine (20 ml of each), dried over magnesium sulphate,filtered and rotary evaporated to an oil which was dissolved in asolution of methylamine in methanol (120 ml, 7.5M) and incubated at roomtemperature for 24 hours. The solution was filtered and the filtrate wasevaporated to an oil which was redissolved in the minimum volume ofpetroleum (b.p. 60°-80° C.)/ethyl acetate (3/2) and applied to a silicacolumn. Elution with the same solvent gave the desired product (2.3 g)as a white foam which was used directly in the next step without furthercharacterisation.

d) Preparation of Reagent A1

The product from step c) above (2.28 g) was dissolved in dry pyridine(20 ml) and succinic anhydride (0.55 g) and DMAP (0.4 g) were added. Themixture was swirled to dissolve the solids and then left at roomtemperature for 16 hours. The solvent was removed by rotary evaporationand residual pyridine was removed by repeated co-evaporation withtoluene. The residue was partitioned between ethyl acetate and 10% (w/v)citric acid (50 ml of each). The organic layer was washed with waterthen saturated brine (50 ml of each), dried over magnesium sulphate,filtered and rotary evaporated to an oil which was redissolved in theminimum volume of dichloromethane/methanol (19/1) and applied to asilica column. Elution with the same solvent gave the title compound asa colourless glass (1.98 g, 71.4%).

¹³ C-NMR: CH₂ groups of OCCH₂ CH₂ CO: 28.790 ppm and 28.939 ppm CH₃ OAr:54.988 ppm CH₃ OCH×2: 55.251 ppm CH₃ OAr: 56.039 ppm CHODMT and CHOCO:75.518 ppm and 76.424 ppm quat C of DMT: 106.572 ppm and 87.194 ppmCHOCH₃ : 106.572 and 109.102 ppm ArCH : 113.245 ppm, 127.821 ppm,128.337 ppm, 129.160 ppm, 130.250 ppm and 130.280 ppm Ar quat C: 139.893ppm, 136.035 ppm, 144.855 ppm and 158.859 ppm COO: 170.879 ppm COO:177.540 ppm

e) Preparation of: ##STR32##

The product from step d) (1.881 g) was dissolved in dry dichloromethane(5 ml) and N,N'-dicyclohexylcarbodiimide (0.384 g, 0.5 meq) was added.The solid was dissolved by swirling the flask, which was then left at20° C. for one hour. Dicyclohexylurea was filtered off and washed withdichloromethane (3×2 ml). The filtrate and washings were combined androtary evaporated. To this was added a solution of the product fromstep 1) (0.708 g, 1.27 meq) in dry pyridine (6 ml). When dissolution wascomplete, the flask was left at 20° C. for thirty nine hours, and thenrotary evaporated. Residual pyridine was removed by repeatedco-evaporation with toluene. The residual oil was dissolved in theminimum volume of dichloromethane:methanol (96:4), and applied to asilica column. Elution with the same solvent gave the title compound asa yellow foam (0.764 g, 63.4%)

¹ H NMR δ (CDCl₃): 2.6-2.8 (4H, m, 2×COCH₂); 3.25-3.5 (12H, 4s,4×--OCH₃); 3.8 (6H, s, 2×ArOCH₃); 4.0 (¹ H, DMT--OCH); 4.35 (¹ H, m,CHOCH₃); 4.45 (1H, m, CHOCH₃); 4.8 (¹ H, m, CHOH); 5.0 (2H, m, 2×CHOCO);5.15 (2H, m, 2×CHOCH₃); 6.9 (4H, m, aromatics); 7.1-7.55 (9H, m,aromatics).

f) Preparation of Reagent A3: ##STR33##

The product from Step e) (0.5 g, 0.702 mmol) was dissolved in drydichloromethane (15 ml) containing dry diisopropylethylamine (0.495ml,2.81 mmol) and the solution was stirred under a stream of dry argonwhile 2-cyanoethyl-N,N-diisopropylaminochlorophosphine (0.189 ml, 0.843mmol) was added dropwise. The solution was stirred under argon at roomtemperature for a further thirty minutes when TLC indichloromethane:triethylamine (19:1) showed there to be no startingmaterial present. The reaction was quenched by the addition of drymethanol (5 ml), and the solution was diluted with ethyl acetate (200ml). This solution was washed with brine (3×200 ml) and water (200 ml).The organic layer was separated, dried (MgSO₄), filtered and evaporatedto a gum which was redissolved in the minimum volume ofdichloromethane:hexane:triethylamine (42:55:3) and applied to a silicacolumn. Elution with the same solvent followed by elution withdichloromethane:triethylamine (19:1) gave the title compound as acolourless glass (0.52 g, 81.3%)

¹ H NMR δ (CDCl₃): 1.1-1.3 (14H, m, 2×CH(CH₃)₂ ; 2.5 (2H, m, CH₂ CN);2.6-2.8 (4H, m 2×COCH₂); 3.2-3.6 (14H, m 4×--OCH₃ and CH₂ OP); 3.8 (6H,s, 2×ArOCH₃); 4.0 (¹ H, m, DMT--OCH); 4.3 (1H, m, CHOCH₃); 4.4-4.6 (2H,m, CHOCH₃ and CHOP); 5.0 (2H, m, 2×CHOCO); 5.2 (2H, m, 2×CHOCH₃); 6.9(4H, m, aromatics); 7.1-7.5 (9H, m, aromatics).

EXAMPLE 6

The method of Example 2) was repeated to synthesise twooligodeoxyribonucleotides bound to a solid support, except that ReagentA3 was used, dissolved in anhydrous acetonitrile to a concentration of0.1M, in place of Reagent A1.

The two oligonucleotides bound to a solid support by a cleavable linkare illustrated by the formula given in Example 2), wherein --L'-- is acleavable linker of formula: ##STR34##

The two oligonucleotides found after step d) in the method of theinvention were found to be identical to oligonucleotides 1) and 2)described in Example 2), demonstrating scission of the cleavable linkand generation of the desired products.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 2                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       TTTTTTTTTT10                                                                  (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       TTTTTTTTTTTTCGA15                                                             __________________________________________________________________________

We claim:
 1. A compound of formula (3): ##STR35## wherein one or both ofA¹ and A² is a divalent group of the formula (a): ##STR36## in which R¹and R² are each independently H, optionally substituted alkyl,optionally substituted alkoxy, optionally substituted aryloxy, or anelectron withdrawing group; Y is CH₂ ; CH₂ CH₂, NH, S or O; E is anorganic spacer group; and any remaining group represented by A¹ or A² isof the formula (b): ##STR37## wherein: n has a value of from 1 to 5; thecarbon atom marked with an asterisk is attached to an oxygen atom shownin Formula (3); and each R³ independently represents H or optionallysubstituted alkyl; Z¹ is an acid labile protecting group; and --O--PA isa phosphoramidite group, a phosphate ester group or a H-phosphonategroup.
 2. A compound comprising two or more oligonucleotides linked by agroup or groups containing a cleavable linker moiety of formula##STR38## wherein A¹, E and A² are as defined in claim
 1. 3. A compoundaccording to claim 1 wherein A¹ and A² are both of formula (a).
 4. Acompound according to claim 1 wherein A¹ is the group of formula (a) andA² is the group of formula (b).
 5. A compound according to any of claim1 wherein Y is O or CH₂.
 6. A compound according to claim 1 wherein E isan alkyl, aryl or aralkyl spacer group, optionally interrupted by anether, thioether, amino or amido group.
 7. A compound according to claim6 wherein E is optionally substituted phenylene or C₂₋₆ -alkylene.
 8. Acompound according to claim 1 wherein R¹ and R² are each independentlyH, alkyl or alkoxy.
 9. A compound according to claim 1 wherein R³ isindependently C₁₋₄ -alkyl or H.
 10. A compound according to claim 1wherein Z¹ is dimethoxytrityl.
 11. A compound according to claim 1wherein O--PA is a phosphoramidite of formula ##STR39## wherein R₅ andR₆ are each independently optionally substituted alkyl, optionallysubstituted aralkyl, cocyloalkyl and cycloalkyalkyl containing up to 10carbon atoms, or taken together with the nitrogen atom to which they areattached form an optionally substituted pyrollidine or piperidine ringor R₅ and R₆ when taken together with the nitrogen atom to which theyare attached form a saturated nitrogen heterocycle which optionallyincludes one or more additional hetero atom(s) selected from the groupconsisting of nitrogen, oxygen and sulphur; and R₇ represents a hydrogenor a protecting group.
 12. A compound according to claim 1 wherein thegroup of formula (a) is where Y is O and R¹ and R² are both eitherhydrogen or methoxy; and the group of formula (b) is --*CH₂ CH₂ --SO₂--CH₂ --CH₂.
 13. A compound according to claim 1 wherein A¹ and A² areboth of formula (a), Y is O, R¹ and R² are both hydrogen or methoxy, Eis --CH₂ CH₂ --, Z¹ is dimethoxytrityl, and O--PA is a phosphoramiditeof formula ##STR40## wherein R₅ and R₆ are each iso-propyl and R₇ is2-cyanoethyl.
 14. A compound according to claims 1 wherein A¹ is offormula (a) in which Y is O and R¹ and R² are both hydrogen or methoxyand A² is --*CH₂ CH₂ --SO₂ --CH₂ --CH₂ --, Z¹ is dimethoxytrityl, andO--PA is a phosphoramidite of formula ##STR41## wherein R⁵ and R⁶ areboth iso-propyl and R is 2-cyanoethyl.